Vascular dysfunction induced by AGE is mediated by VEGF via mechanisms involving reactive oxygen species, guanylate cyclase, and protein kinase C.

OBJECTIVE: These experiments were designed to elucidate mechanisms mediating vascular dysfunction induced by advanced glycation end products (AGEs). METHODS: Skin chambers were mounted on the backs of Sprague-Dawley rats and 1 week later, granulation tissue that formed in the bottom of the chamber was exposed twice daily for 7 days to glycated rat serum albumin in the presence and absence of inhibitors of reactive oxygen intermediates, nitric oxide synthase and guanylate cyclase, protein kinase C (PKC), and a neutralizing vascular endothelial growth factor (VEGF) antibody. Vascular (125)I-albumin clearance and blood flow were quantified by use of a double isotope-dilution technique and radiolabeled microspheres, respectively. RESULTS: Albumin permeation and blood flow were increased dose-dependently to a maximum of 2 to 3 times controls by increasing the extent of glucose modification, the concentration, or the duration of exposure to glycated albumin. These increases were significantly attenuated by probucol and superoxide dismutase; N(G)-nitro-L-arginine-methyl ester (L-NAME), a nitric oxide synthase inhibitor; LY83583, a guanylate cyclase inhibitor; and LY333531, a beta-isoform-selective protein kinase C inhibitor. A neutralizing VEGF monoclonal antibody also markedly attenuated the permeability and blood flow increases induced by glycated albumin. CONCLUSIONS: These observations indicate potentially important roles for oxygen free-radicals and nitric oxide in mediating permeability and blood flow changes induced by glycated proteins via mechanisms involving increased protein kinase C activity and VEGF production. Striking similarities in the mechanism by which hyperglycemia and glycated proteins induce vascular dysfunction suggest that a common pathway mediates effects of these different metabolic imbalances on vascular dysfunction.

Role for nitric oxide in the hyperpermeability and hemodynamic changes induced by intravenous VEGF.

PURPOSE: To explore the effects of brief intravenous (IV) infusion of vascular endothelial growth factor (VEGF) on vascular albumin permeability, blood flow, and vascular conductance (blood flow normalized to arterial blood pressure) in ocular tissues and brain and to assess the role of nitric oxide in mediating these changes. METHODS: A quantitative, double-tracer, radiolabeled albumin permeation method was combined with radiolabeled microspheres for assessment of changes in vascular permeability and blood flow, respectively, induced in ocular tissues by IV infusion of recombinant human VEGF165 for 20 minutes (80-450 picomoles/kg body weight). An inhibitor of nitric oxide synthase (NOS), NG-monomethyl-L-arginine (L-NMMA; 50 micromoles/kg body weight infused simultaneously with VEGF), was used to explore the role of nitric oxide in mediating the vascular changes induced by VEGF. RESULTS: Infusion of VEGF165 in thiopental-anesthetized rats dose-dependently increased 125I albumin permeation in the retina, anterior uvea, and choroid/sclera and in brain, aorta, lung, kidney, small intestine, and peripheral nerve. Mean arterial blood pressure, cardiac output, and stroke volume were decreased only at the highest dose of VEGF, whereas heart rate remained unchanged. Blood flow was increased in the anterior uvea, and vascular conductance was increased in retina, anterior uvea, choroid/sclera, and brain at the highest dose of VEGF. The NOS inhibitor, L-NMMA, blocked VEGF-induced vascular hyperpermeability in all ocular and nonocular tissues, prevented the increase in vascular conductance in all ocular tissues, and blocked the decrease in mean arterial blood pressure, cardiac output, and stroke volume. Infusion of L-NMMA alone decreased vascular conductance in choroid/sclera and kidney, slightly increased mean arterial blood pressure, and in general, did not affect 125I-albumin permeation. (L-NMMA slightly decreased albumin permeation in the retina and increased it in the brain.) CONCLUSIONS: Intravenous infusion of VEGF can acutely impair endothelial cell barrier functional integrity and relax resistance arterioles in ocular tissues and brain through a mechanism involving activation of NOS.

Role for heparin-binding growth factors in glucose-induced vascular dysfunction.

Vascular hyperpermeability and excessive neovascularization are hallmarks of early and late vascular endothelial cell dysfunction induced by diabetes. Vascular endothelial growth factor (VEGF) appears to be an important mediator for these early and late vascular changes. We reported previously, using skin chambers mounted on backs of SD rats, that neutralizing antibodies directed against VEGF blocked vascular permeability and blood flow changes induced by elevated tissue glucose and sorbitol levels in a dosage-dependent manner. We report in this study, using the same skin chamber model and neutralizing antibodies directed against basic fibroblast growth factor (FGF-2), that another member of the heparin-binding growth factor family also mediates glucose- and sorbitol-induced vascular permeability and blood flow increases. In addition, we show that 1) TBC1635, a novel heparin-binding growth factor antagonist, blocks the vascular hyperpermeability and blood flow increases induced by elevated tissue levels of glucose and sorbitol and by topical application of VEGF and FGF-2 to granulation tissue in skin chambers, and 2) suramin, a commercially available growth factor antagonist, blocks glucose-induced vascular dysfunction. These results suggest an early role for heparin-binding growth factors in the vascular dysfunction caused by excessive glucose metabolism, possibly via the sorbitol pathway.

Diabetes impairs sciatic nerve hyperemia induced by surgical trauma: implications for diabetic neuropathy.

The most widely used methods to assess nerve blood flow in diabetics rats are hydrogen clearance polarography and laser Doppler flowmetry, techniques requiring surgical exposure of the nerve. In these experiments, we examined the hypothesis that the trauma of surgical exposure introduces an important and hitherto largely unrecognized variable that could account for discordant reports on nerve blood flow changes induced by diabetes. We used the noninvasive (for sciatic nerve) reference sample microsphere method to quantify sciatic nerve blood flow in unexposed va. surgically exposed nerves in rats with streptozotocin-induced diabetes (at different temperatures and after curarization) and in unexposed vs. surgically exposed nerves in galactose-fed rats. Baseline resting blood flow in unexposed nerves in both animal models of diabetes was either normal or increased (but was decreased in diabetic rats given d-tubocurarine). Furthermore, the normal brisk hyperemic nerve blood flow response to the minimal trauma associated with surgical exposure of the nerve was markedly impaired in diabetic and in galactose-fed rats. Normalization of the blood flow response to trauma in galactose-fed rats by an aldose reductase inhibitor suggests that the impairment is linked to increased polyol pathway metabolism. These findings 1) confirm our previous findings that sciatic nerve blood flow in diabetic rats is increased or unchanged in unexposed nerves, while also confirming reports that in surgically exposed nerves blood flow is higher in control than in diabetic rats, and 2) indicate that blood flows in surgically exposed nerves are largely a measure of vascular responses to injury rather than (patho)physiological blood flow in undisturbed nerves.

Increased sciatic nerve blood flow in diabetic rats: assessment by "molecular" vs. particulate microspheres.

Sciatic nerve blood flow in diabetic rats in typically increased or unchanged when assessed by the reference sample microsphere method in our laboratory. In contrast, blood flow is generally reported to be decreased approximately 50% when assessed with laser Doppler flowmetry or hydrogen clearance polarography. To address concerns that increased blood flow observed with microspheres might be anomalous because of their particulate nature and/or because insufficient numbers of microspheres are captured in the nerve, a plasma-soluble "molecular microsphere" ([3H]desmethylimipramine, mol wt = 266) and 11.3-micron 153Gd-labeled microspheres were injected sequentially to assess blood flow in rats with streptozotocin diabetes of 2-4 wk duration. Nerve blood flows in diabetic rats were increased 1.5- to 2-fold (vs. control rats) with both tracers; these increases were prevented by tolrestat, an inhibitor of aldose reductase. These observations indicate that blood flow in sciatic nerve (like that in retina and kidney) is increased early after the onset of diabetes and is 1) demonstrable with a plasma-soluble tracer as well as with particulate microspheres and 2) linked to increased metabolism of glucose via the sorbitol pathway.

TNF-alpha causes reversible in vivo systemic vascular barrier dysfunction via NO-dependent and -independent mechanisms.

Tumor necrosis factor (TNF-alpha) and nitric oxide (NO) are important vasoactive mediators of septic shock. This study used a well-characterized quantitative permeation method to examine the effect of TNF-alpha and NO on systemic vascular barrier function in vivo, without confounding endotoxemia, hypotension, or organ damage. Our results showed 1) TNF-alpha reversibly increased albumin permeation in the systemic vasculature (e.g., lung, liver, brain, etc.); 2) TNF-alpha did not affect hemodynamics or blood flow or cause significant tissue injury; 3) pulmonary vascular barrier dysfunction was associated with increased lung water content and impaired oxygenation; 4) TNF-alpha caused inducible nitric oxide synthase (iNOS) mRNA expression in the lung and increased in vivo NO production; 5) selective inhibition of iNOS with aminoguanidine prevented TNF-alpha-induced lung and liver vascular barrier dysfunction; 6) aminoguanidine prevented increased tissue water content in TNF-alpha-treated lungs and improved oxygenation; and 7) nonselective inhibition of NOS with NG-monomethly-L-arginine increased vascular permeation in control lungs and caused severe lung injury in TNF-alpha-treated animals. We conclude that 1) TNF-alpha reversibly impairs vascular barrier integrity through NO-dependent and -independent mechanisms; 2) nonselective NOS inhibition increased vascular barrier dysfunction and caused severe lung injury, whereas selective inhibition of iNOS prevented impaired endothelial barrier integrity and pulmonary dysfunction; and 3) selective inhibition of iNOS may be beneficial in treating increased vascular permeability that complicates endotoxemia and cytokine immunotherapy.